: Cells respond to stress in part by altering gene expression. A critical control point for regulating gene expression in eukaryotic cells is during mRNA transcription by RNA polymerase II (PolII). Recently, non-coding RNA molecules (ncRNAs) have been found to regulate mRNA transcription. Mouse B2 RNA and human Alu RNA are two such ncRNAs; they function as repressers of mRNA transcription by binding directly to Pol II in response to heat shock, a widely used model system for studying the cellular stress response. The proposed studies will investigate how B2 RNA and Alu RNA define the transcriptional program that occurs as cells respond to and then recover from heat shock, how these ncRNAs are regulated in response to heat shock, and their structural and sequence determinants that allow binding to Pol II and transcriptional repression. The specific aims of the proposal are: 1) To determine the molecular events that occur at repressed genes as cells respond to and recover from heat shock, 2) To understand the ncRNA and protein components involved in forming Pol ll/ncRNA complexes, 3) To understand the mechanism of transcriptional repression by ncRNAs that bind Pol II, and 4) To identify factors that derepress transcription in the presence of these ncRNAs. The specific aims utilize in vitro experiments and cell- based assays. The in vitro experiments employ a purified Pol II transcription system and nuclear extracts. The cell-based experiments utilize methods such as chromatin immunoprecipitations and a novel variation of this technique, antisense technologies, and microarrays. The proposed experiments will likely reveal both novel mechanisms of transcriptional regulation not observed with protein regulators and the broad potential for regulation of Pol II by yet unidentified ncRNAs, as well as generate the first comprehensive view of transcriptional repression in response to heat shock in both mouse and human cells. Relevance to public health: Controlling gene expression is essential to growth, development, and sustained life. The proper regulation of transcription (making RNA from DNA) is essential to maintaining normal pathways of cell growth and differentiation, thereby avoiding the rampant cell proliferation observed in tumors. Completion of these studies will contribute to discerning how transcription is regulated during cellular stress, which is critical for understanding abnormalities in gene expression associated with diseases and deleterious environmental states.